Synergy between low molecular weight heparin and platelet aggregation inhibitors, providing a combination therapy for the prevention and treatment of various thromboembolic disorders

ABSTRACT

This invention is directed to a combination therapy comprising the administration of a low molecular weight heparin such as tinzaparin and a platelet GPIIb/IIIa antagonist such as roxifiban for treating, preventing and reducing the risk of thromboembolic disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/123,820, filed Mar. 11, 1999.

FIELD OF THE INVENTION

[0002] This invention is directed to a combination therapy comprisingthe administration of a low molecular weight heparin such as tinzaparinand a platelet GPIIb/IIIa antagonist such as roxifiban for treating,preventing and reducing the risk of thromboembolic disorders.

BACKGROUND OF THE INVENTION

[0003] Thromboembolic diseases, including stable and unstable anginapectoris, myocardial infarction, stroke and lung embolism, are the majorcause of disability and mortality in most developed countries. Plateletglycoprotein IIb/IIIa (GPIIb/IIIa) is the main platelet receptor forfibrinogen and other adhesive glycoproteins, including fibronectin,vitronectin and von Willebrand factor. Interference of ligand bindingwith this receptor has been proven beneficial in animal models ofthromboembolic disease (Coller, B. S. GPIIb/IIIa Antagonists:Pathophysiologic and Therapeutic Insights from Studies of C7E3 FAB.Thromb. Haemost. 78: 1, 730-735, 1997), and in limited studies involvinghuman subjects (White, H. D. Unmet Therapeutic Needs in the Managementof Acute Ixchemia. Am. J. Cardiol. 80: 4A, 2B-10B, 1997; Tcheng, J. E.Glycoprotein IIb/IIIa Receptor Inhibitors: Putting EPIC, IMPACT II,RESTORE, and EPILOG Trials Into Perspective. Am. J. Cardiol. 78: 3A,35-40, 1996).

[0004] The integrin GPIIb/IIIa, also referred to as the plateletfibrinogen receptor, is the membrane protein mediating plateletaggregation. GPIIb/IIIa in activated platelets is known to bind foursoluble RGD containing adhesive proteins, namely fibrinogen, vonWillebrand factor, fibronectin, and vitronectin. The term “RGD” refersto the amino acid sequence Arg-Gly-Asp. The binding of fibrinogen andvon Willebrand factor to GPIIb/IIIa causes platelets to aggregate.GPIIb/IIIa antagonists represent an important new approach foranti-platelet therapy for the treatment of thromboembolic disorders.

[0005] Platelet activation and aggregation are involved in unstableangina and acute myocardial infarction, in reocclusion followingthrombolytic therapy and angioplasty, in transient ischemic attacks andin a variety of other vaso-occlusive disorders. When a blood vessel isdamaged either by acute intervention such as angioplasty, or morechronically by the pathophysiological processes of atherosclerosis,platelets are activated and adhere to the disrupted surface and to eachother. This activation, adherence and aggregation may lead to occlusivethrombus formation in the lumen of the blood vessel. Since the bindingof fibrinogen to an activated membrane-bound glycoprotein complex(GPIIb/IIIa) is an obligatory component of normal aggregation,GPIIb/IIIa is an attractive target for an antithrombotic agent.

[0006] Antiplatelet therapy has been used in a wide variety ofcardiovascular disease states and in conjunction with interventionaltherapy such as coronary artery or peripheral bypass grafting, cardiacvalve replacement, and percutaneous transluminal coronary angioplasty(PTCA). Available drugs, such as aspirin and ticlopidine (TICLID®), haveshown efficacy in syndromes involving vascular occlusion, presumably dueto sustained inhibition of platelet function. However, the inhibitoryeffects of aspirin and ticlopidine are dependent upon the agonist, whichactivates the platelet. For example, aspirin is effective in blockingplatelet aggregation induced by agonists such as collagen that aredependent upon the cyclooxygenase pathway. It is however, less effectiveagainst concentrations of thrombin which can act by cyclooxygenaseindependent pathways. Likewise, the inhibitory effects of tidopidine,which inhibits ADP induced platelet aggregation, can be overcome bycombinations of agonists.

[0007] The effect of heparin on blood coagulation is exploitedclinically as an anticoagulant and antithrombotic drug. Heparin belongsto the group of polysaccharides known as glycosaminoglycans (GAGs), andis composed of alternating 1-4-linked hexuronic acid and D-glucosamine.Both the hexauronic acid and the glucosamine residues are sulfated in acomplex pattern resulting in extensive structural variability. Becauseof its high negative-charge density, heparin is able to interact withclusters of basic amino acids on numerous proteins and cell membranes,such as coagulation proteinases, serine protease inhibitors, growthfactors, lipoprotein and hepatic lipase, apolipoproteins B and E,adhesive matrix proteins, platelets, and endothelial cells.

[0008] Low molecular weight heparins (LMWHs), obtained from standardunfractionated heparin (UFH), are as effective as standardunfractionated heparin for prophylaxis and treatment of venousthromboembolism and have fewer side effects (Schafer A. I. Lowmolecular-weight heparin for venous thromboembolism. Hospital Practice,Jan. 15, 1997, pp. 99-106). The current available low molecular weightheparins include, for example, tinzaparin, certoparin, parnaparin,nadroparin, ardeparin, enoxaparin, reviparin and dalteparin (fragmin).

[0009] A combination therapy comprising the administration of aGPIIb/IIIa antagonist with either aspirin or a low molecular weightheparin is useful in the treatment of thrombotic disorders includingatherosclerotic arterial disease, valvular heart disease,cerebrovascular disease such as stroke, atrial fibrillation, coronaryartery disease such as myocardial infarction and unstable angina,coronary artery bypass grafts, peripheral vascular disease,thromboembolic complications of prosthetic cardiovascular devices suchheart valves and vascular grafts. These combinations are also expectedto be useful when coupled with endovascular stenting procedures, such aspercutaneous transluminal coronary angioplasty, to prevent subsequentarterial thrombus formation and reocclusion.

[0010] A number of criteria must be considered when contemplating acombination therapy: (a) each agent demonstrates significant clinicalbenefits in various thromboembolic disorders; (b) both agents act atdifferent mechanistic levels and with different capacities; (c)combination of the agents at adjusted doses could improve efficacyand/or safety. In that regard, a wealth of clinical experience exists,especially for UFH and intravenous platelet GPIIb/IIIa antagonists inacute coronary syndromes (ACS). The potential clinical benefit of theplatelet GPIIb/IIIa antagonist abciximab in ACS was demonstrated in thepivotal “Evaluation of c7E3 Fab in the Prevention of IschemicComplications” (EPIC) and “Evaluation in Percutaneous transluminalcoronary angioplasty to Improve Long-term Outcome with abciximabGPIIb/IIIa blockade” (EPILOG) trials. In the EPIC trial, there wassignificant excess bleeding that occurred when UFH was used in its fulldose with abciximab leading to the EPILOG trial, where a reduced dose ofUFH was used. The lower UFH dosage led to an improved safety profilewithout compromising the efficacy observed in the EPIC trial. Similarly,the “Platelet Receptor Inhibition in Ischemic Syndrome Management”(PRISM) and the “PRISM in Patients Limited by Unstable Signs andSymptoms” (PRISM-PLUS) trials evaluated whether administration ofaspirin (PRISM) or UFH plus tirofiban (PRISM-PLUS) would improveclinical outcomes in the management of unstable angina. The controlgroup in PRISM-PLUS received intravenous UFH only. Tirofiban plus UFHwas significantly more effective than UFH alone in reducing theincidence of death, myocardial infarction, or refractory ischemia within7 days after randomization. This finding suggested improved efficacyfollowing administration of the combination of UFH and a GPIIb/IIIaantagonist. Other trials of GPIIb/IIIa antagonists with adjusted-doseUFH are currently under investigation. The advantages of LMWHs over UFHare significant (as described hereinafter), therefore the combination ofa LMWH with a platelet GPIIb/IIIa receptor antagonist has greatpotential benefits when combined at the right dose regimens.

SUMMARY OF THE INVENTION

[0011] One object of the present invention is to provide a method oftreating thrombosis in a mammal comprising: administering to said mammalthe combination in a therapeutically effective amount of (i) aGPIIb/IIIa antagonist selected from the group consisting of abciximab,eptifibatide, tirofiban, amifiban, lefradafiban, sibrafiban, orbofiban,xemilofiban, a compound of the formula (A):

[0012] and a compound of the formula (B) (roxifiban):

[0013] and (ii) aspirin, or the GPIIb/IIIa antagonist (i) and (iii) alow molecular weight heparin selected from the group consisting oftinzaparin, certoparin, parnaparin, nadroparin, ardeparin, enoxaparin,reviparin and dalteparin, wherein at least one of the antagonist andaspirin, or at least one of the antagonist and the low molecular weightheparin, is administered in a subtherapeutic amount.

[0014] Another object of the present invention is to provide a method oftreating thrombosis in a mammal wherein the combination of (i) and (ii)or (iii) above are administered in amounts to provide a synergisticeffect.

[0015] Another object of the present invention is to provide a method oftreating thrombosis in a mammal by administering to said mammal thecombination in a therapeutically effective amount of tissue plasminogenactivator (“TPA”), and a GPIIb/IIIa antagonist compound of the formula

[0016] wherein at least one of said agents is administered in asubtherapeutic dose.

[0017] Another object of the present invention is to provide a methodfor treating a condition selected from the group: acute coronaryischemic syndrome, thrombosis, thromboembolism, thrombic occlusion andreclusion, restenosis, transient ischemic attack, and first orsubsequent thrombotic stroke comprising the subcutaneous administrationof a sub-therapeutic dose of a low molecular weight heparin incombination with a sub-therapeutic dose of a platelet GPIIb/IIIaantagonist to a mammal at risk of developing one or more of saidconditions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1. Graph illustrating the effect of roxifiban on theantiplatelet efficacy of tinzaparin in thrombin-induced human plateletaggregation.

[0019]FIG. 2. Graph illustrating the syngeristic antiplatelet effects oftinzaparin and roxifiban on thrombin-induced platelet aggregation.

[0020]FIG. 3. Graph illustrating carotid blood flow versus time forsaline vehicle, aspirin alone, a GPIIb/IIIa antagonist alone, and acombination of aspirin and the same GPIIb/IIIa antagonist.

[0021]FIG. 4. Graph illustrating carotid blood flow versus time forsaline vehicle, dalteparin (fragmin) alone, a GPIIb/IIIa antagonistalone, and a combination of dalteparin and the same GPIIb/IIIaantagonist.

[0022]FIG. 5. Schematic illustration of the instrumentation utilized inthromboelastography and the parameters of the measurements.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The combinations of a GPIIb/IIIa antagonist with either aspirinor a low molecular weight heparin is useful in the treatment ofthrombotic disorders including atherosclerotic arterial disease,valvular heart disease, cerebrovascular disease such as stroke, atrialfibrillation, coronary artery disease such as myocardial infarction andunstable angina, coronary artery bypass grafts, peripheral vasculardisease, thromboembolic complications of prosthetic cardiovasculardevices such heart valves and vascular grafts. These combinations arealso expected to be useful when coupled with endovascular stentingprocedures, such as percutaneous transluminal coronary angioplasty, toprevent subsequent arterial thrombus formation and reocclusion. Alsouseful in the treatment of thrombosis is the combination in atherapeutically effective amount of tissue plasminogen activator and theGPIIb/IIIa antagonist compound (B), described herein.

[0024] Aspirin useful in the combination of the present invention iscommercially available and well known in the art. Low molecular weightheparins such as dalteparin (fragmin) useful in the combination of thepresent invention are also commercially available and well known in theart. Preferred GPIIb/IIIa antagonist compounds useful herein, as well astheir preparation, are described in WO 95/14683, U.S. Pat. No. 5,849,736(the contents of which are herein incorporated by reference). Preferredcompounds described therein and the preparation thereof have theformula:

[0025] Compound (A):

[0026] and Compound (B):

[0027] other salts and prodrug forms of these compounds are alsopreferred.

[0028] An embodiment of the present invention describes a method fortreating a condition selected from the group: acute coronary ischemicsyndrome, thrombosis, thromboembolism, thrombic occlusion and reclusion,restenosis, transient ischemic attack, and first or subsequentthrombotic stroke comprising administration of a sub-therapeutic dose ofa low molecular weight heparin and a sub-therapeutic dose of a plateletGPIIb/IIIa antagonist to a mammal at risk of developing one or more ofsaid conditions. In a preferred embodiment of the present invention theplatelet GPIIb/IIIa antagonist is roxifiban. In a preferred embodimentof the present invention the low molecular weight heparin is tinzaparin.In a preferred embodiment of the present invention the administration issubcutaneous.

[0029] Another embodiment of the present invention describes apharmaceutical composition comprising a platelet GPIIb/IIIa antagonistand a low molecular weight heparin and a pharmaceutically acceptablecarrier. In a preferred embodiment of the present invention the plateletGPIIb/IIIa antagonist is roxifiban. In a preferred embodiment of thepresent invention the low molecular weight heparin is tinzaparin.

[0030] Another embodiment of the present invention describes a kitcomprising a platelet GPIIb/IIIa antagonist and a low molecular weightheparin for subcutaneous injection.

[0031] Another embodiment of the present invention describes a method oftreating thrombosis in a mammal comprising administering to said mammala combination of: (i) a subtherapeutic dose of GPIIb/IIIa antagonistselected from the group consisting of abciximab, eptifibatide,tirofiban, lamifiban, lefradafiban, sibrafiban, orbofiban, xemilofiban,a compound of the formula (A):

[0032] and a compound of the formula (B):

[0033] and (ii) aspirin or (iii) a low molecular weight heparin selectedfrom the group consisting of tinzaparin, certoparin, parnaparin,nadroparin, ardeparin, enoxaparin, reviparin, reviparin and dalteparin.In a preferred embodiment of the present invention the combination of(i) and (ii) or (iii) provides a synergistic effect. In a preferredembodiment of the present invention the combination administered is acombination of the GPIIb/IIIa antagonist and a low molecular weightheparin. In a preferred embodiment of the present invention theGPIIb/IIIa antagonist is administered in a subtherapeutic amount. In apreferred embodiment of the present invention the GPIIb/IIIa antagonistis compound A or compound B. In a preferred embodiment of the presentinvention the low molecular weight heparin is dalteparin. In a morepreferred embodiment of the present invention the GPIIb/IIIa antagonistis compound A or compound B and the low molecular weight heparin isdalteparin.

[0034] Another embodiment of the present invention use of a combinationof: (i) a subtherapeutic dose of GPIIb/IIIa antagonist selected from thegroup consisting of abciximab, eptifibatide, tirofiban, lamifiban,lefradafiban, sibrafiban, orbofiban, xemilofiban, a compound of theformula (A):

[0035] and a compound of the formula (B):

[0036] and (ii) aspirin or (iii) a low molecular weight heparin selectedfrom the group consisting of tinzaparin, certoparin, parnaparin,nadroparin, ardeparin, enoxaparin, reviparin, and dalteparin (fragmin)for the manufacture of a medicament for the treatment of thrombosis.

[0037] Another embodiment of the present invention describes a method oftreating thrombosis in a mammal comprising administering to said mammal(i) tissue plasminogen activator and (ii) a compound of the formula:

[0038] wherein at least one of (i) or (ii) is administered in asubtherapeutic amount.

[0039] Specific examples of useful GPIIb/IIIa antagonist compounds areroxifiban, abciximab, eptifibatide, tirofiban, lamifiban, lefradafiban,sibrafiban (Ro-48-3657), orbofiban and xemilofiban described in thepaper of Graul et al. and Scarborough (Graul A, Martel A M and CastanerJ. Xemilifiban; Drugs of the Future 22: 508-517, 1997; Scarborough R M;Eptifibatide. Drugs of the Future 23: 585-590, 1998). Of these,roxifiban, lamifiban, lefradafiban, sibrafiban, orbofiban andxemilofiban are preferred. Others will be readily apparent to thoseskilled in the art.

[0040] The effects of the combination of tinzaparin and the plateletGPIIb/IIIa antagonist roxifiban on clot formation and platelet-plateletaggregate formation were studied using thromboelastography (TEG) forplatelet-fibrin clot dynamic studies, and light-transmittanceaggregometry (LTA) for platelet-platelet interaction studies. The effectof GPIIb/IIIa blockade by roxifiban at sub-therapeutic concentration onthe IC₅₀ tinzaparin demonstrated a significant, 2-fold shift of theIC₅₀. LTA was also utilized to study this interaction. Roxifiban has asa steep dose-response relationship like any other GPIIb/IIIa antagonist.A significant enhancement of tinzaparin's efficacy was shown whenroxifiban was combined at sub-therapeutic concentration with differentconcentrations of tinzaparin (FIG. 1), suggesting a synergisticenhancement of the anti-platelet efficacy of roxifiban by tinzaparin andvice versa (FIG. 2). Combining potent GPIIb/IIIa antagonists such asabciximab with LMWHs results in a similar synergistic interaction.Tissue factor pathway inhibitor (TFPI) can inhibit tissue factor (TF),modulate integrin, down-regulate pro-inflammatory stimuli, providecytoprotective, anti-platelet effects, modulate Xa, and leukocytefunctions. Different LMWHs administered intravenously to non-humanprimates at equivalent anti-Xa units demonstrated different levels ofactivity in releasing free TFPI compared to other LMWHs. Additionally,tinzaparin inhibited TF-induced expression of platelet P-selectin, whichis important in the modulation of leukocyte-platelet binding andinduction of inflammatory stimuli.

[0041] The interaction between platelet GPIIb/IIIa receptor antagonistsand LMWHs may have tremendous clinical implications. LMWHs may enhancethe anti-platelet activity of GPIIb/IIIa antagonists by inhibitingthrombin and Xa. At the same time, GPIIb/IIIa antagonists may potentiatethe anti-coagulant action of LMWHs by blocking fibrinogen binding andaggregation regardless of the activating stimulus; they also contributeto the down-regulation of pro-coagulant activity on the plateletsurface. The combination of reduced dose of an LWMH with a reduced doseof a GPIIb/IIIa antagonist may result in a higher therapeutic index.

[0042] In treating venous thrombosis and, to a certain extent, acuteischemic syndromes, low-molecular-weight heparin (LMWH) has a moreconvenient delivery method (subcutaneous) for outpatient use andimproved efficacy and safety compared with unfractionated heparin.Computerized Thromboelastography (TEG) was used to determine the abilityof platelets and fibrin to augment the shear elastic modules of bloodclots. This augmentation was quantified under conditions of maximalplatelet activation during clot formation accelerated by recombinanthuman tissue factor (TF). A comparative efficacy between LMWHs ondifferent mediator-induced clot retraction in human blood using TEGmeasurements was carried out. These data demonstrated the potency ofdifferent LMWHs in inhibiting various mediator-induced clot formationsunder shear. Tinzaparin demonstrated relatively higher potency ininhibiting TF, lipopolysaccharide (LPS), Xa, and thrombin-induced clotformation under shear. The data suggest a broader efficacy of LMWHcompared with other anticoagulant mechanisms. Under these conditions,platelets significantly enhance clot strength eight folds (relative toplatelet-free fibrin clots). Abciximab and roxifiban inhibitedenhancement of clot strength by affecting the transmission of plateletcontractile force to fibrin via platelet GPIIb/IIIa receptors.Tinzaparin demonstrated high potency in inhibiting clot formation,mediated by TF, Xa, or thrombin. The combination of sub-therapeutictinzaparin and sub-therapeutic roxifiban or Abciximab resulted indistinct synergy in improving antiplatelet and anticoagulant efficacy,mediated by TF, Xa, or thrombin. These data support the usefulness oflow-dose tinzaparin with low-dose GPIIb/IIIa antagonists, such asAbciximab or roxifiban, in the prevention and treatment of differentthromboembolic disorders.

[0043] Compared to unfractionated heparin (UFH), LMWHs exhibit improvedsubcutaneous (SC) bioavailability; lower protein binding; longerhalf-life; variable number of antithrombin III binding sites; variableglycosaminoglycan contents; variable anti-serine protease activities(anti-Xa, anti-IIa, and anti-Xa/anti-IIa ratio); variable potency inreleasing TPFPI; variable levels of vascular endothelial cell bindingkinetics; and hence variable vascular protective effects. For thesereasons, over the last decade LMWHs have increasingly replaced UFH inthe prevention and treatment of venous thromboembolic disorders (VTE).Randomized clinical trials have demonstrated that individual LMWHs usedat optimized dosages are at least as effective and probably safer thanUFH. The convenient once- or twice-daily SC dosing regimen without theneed for monitoring has encouraged the wide use of LMWHs. It is wellestablished that different LMWHs vary in their physical and chemicalproperties due to the differences in their methods of manufacturing.These differences translate into differences in their pharmacodynamicand pharmacokinetic characteristics. The World Health Organization (WHO)and United States Food and Drug Administration (FDA) regard LMWHs asindividual drugs that cannot be used interchangeably.

[0044] As used herein, the phrase “therapeutically effective amount” isintended to include an amount of a combination of compounds claimedeffective to treat thrombosis in a mammal. The combination of compoundsis preferably a synergistic combination. “Synergy”, as described forexample by Chou and Talalay, Adv. Enzyme Regul. 22:27-55 (1984), occurswhen the effect (in this case, an antithrombotic effect) of thecompounds when administered in combination is greater than the additiveeffect of the compounds when each is administered alone as a singleagent. In general, a synergistic effect is most clearly demonstrated atsubtherapeutic amounts of one or more of the combined compounds. Synergycan be in terms of antihypertensive effect, antithrombotic effect, orsome other non-additive beneficial effect of the combination comparedwith the individual components.

[0045] The terms “administered in combination”, “combination”, or“combined” when referring to compounds described herein, means thecompounds or components are administered concurrently to the mammalbeing treated. When administered in combination each compound orcomponent or component may be administered at the same time orsequentially in any order or at different points in time, so as toprovide the desired therapeutic effect. By “subtherapeutic amount”, itis meant that each component when administered to a mammal alone doesnot give the desired therapeutic effect for the disease being treated.

[0046] “Prodrugs”, as the term is used herein, are intended to includeany covalently bonded carriers which release an active parent drug ofthe present invention in vivo when such prodrug is administered to amammalian subject. Since prodrugs are known to enhance numerousdesirable qualities of pharmaceuticals (i.e., solubility,bioavailability, manufacturing, etc.) the compounds of the presentinvention may be delivered in prodrug form. Prodrugs include compoundsof the present invention wherein a hydroxy, amino, or sulfhydryl groupis bonded to any group that, when the prodrug of the present inventionis administered to a mammalian subject, it cleaves to form a freehydroxyl, free amino, or free sulfydryl group, respectively. Examples ofprodrugs include, but are not limited to, acetate, formate, and benzoatederivatives of alcohol and amine functional groups in the compounds ofthe present invention.

Dosage and Formulation

[0047] Combinations of GPIIb/IIIa antagonists and either aspirin or lowmolecular weight heparin, as well as TPA and the GPIIb/IIIa antagonistcompound B, are administered as treatment for thrombosis by any meansthat produces contact of the agents with their site of action, i.e., theGPIIb/IIIa receptor, in the body of a mammal. They can be administeredby any conventional means available for use in conjunction withpharmaceuticals, either as individual therapeutic agents or in acombination of therapeutic agents.

[0048] Dosage forms of compositions suitable for administration containfrom about 1 mg to about 100 mg of active ingredient per unit. In thesepharmaceutical compositions the active ingredient will ordinarily bepresent in an amount of about 0.5-95% by weight based on the totalweight of the composition. The active ingredient can be administeredorally in solid dosage forms, such as capsules, tablets and powders, orin liquid dosage forms, such as elixirs, syrups and suspensions. It canalso be administered parenterally, in sterile liquid dosage forms.

[0049] Gelatin capsules contain the active ingredient and powderedcarriers, such as lactose, starch, cellulose derivatives, magnesiumstearate, stearic acid, and the like. Similar diluents can be used tomake compressed tablets. Both tablets and capsules can be manufacturedas sustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration can contain coloring and flavoring to increasepatient acceptance.

[0050] In general, water, suitable oil, saline, aqueous dextrose(glucose), and related sugar solutions and glycols such as propyleneglycol or polyethylene glycols are suitable carriers for parenteralsolutions. Solutions for parenteral administration preferably contain awater-soluble salt of the active ingredient, suitable stabilizingagents, and if necessary, buffer substances. Antioxidizing agents suchas sodium bisulfite, sodium sulfite, or ascorbic acid, either alone orcombined, are suitable stabilizing agents. Also used are citric acid andits salts, and sodium EDTA. In addition, parenteral solutions cancontain preservatives, such as benzalkonium chloride, methyl- orpropyl-paraben and chlorobutanol. Suitable pharmaceutical carriers aredescribed in Remington's Pharmaceutical Sciences, 19th ed., MackPublishing Company, Easton, Pa., 1995, a standard reference text in thisfield, the contents of which are herein incorporated by reference.

[0051] Useful pharmaceutical dosage-forms for administration of thecompounds of this invention can be illustrated as follows:

Capsules

[0052] A large number of unit capsules can be prepared by fillingstandard two-piece hard gelatin capsules each with 0.1 to 100 mg ofpowdered active ingredient, 150 mg of lactose, 50 mg of cellulose, and 6mg magnesium stearic.

Soft Gelatin Capsules

[0053] A mixture of active ingredient in a digestible oil such assoybean oil, cottonseed oil or olive oil can be prepared and injected bymeans of a positive displacement pump into gelatin to form soft gelatincapsules containing 0.1 to 100 mg of the active ingredient. The capsulesshould then be washed and dried.

Tablets

[0054] A large number of tablets can be prepared by conventionalprocedures so that the dosage unit is 0.1 to 100 mg of activeingredient, 0.2 mg of colloidal silicon dioxide, 5 milligrams ofmagnesium stearate, 275 mg of microcrystalline cellulose, 11 mg ofstarch and 98.8 mg of lactose. Appropriate coatings may be applied toincrease palatability or delay absorption.

Suspension

[0055] An aqueous suspension can be prepared for oral administration sothat each 5 mL contain 0.1 to 100 mg of finely divided activeingredient, 200 mg of sodium carboxymethyl cellulose, 5 mg of sodiumbenzoate, 1.0 g of sorbitol solution, U.S.P., and 0.025 mg of vanillin.

Injectable

[0056] A parenteral composition suitable for administration by injectioncan be prepared by stirring 0.1 to 100 mg by weight of active ingredientin 10% by volume propylene glycol and water. The solution is sterilizedby commonly used techniques.

[0057] The combined compounds of this invention may be formulated suchthat, although the active ingredients are combined in a single dosageunit, the physical contact between the active ingredients is minimized.In order to minimize contact, for example, where the product is orallyadministered, one active ingredient may be enteric coated. By entericcoating one of the active ingredients, it is possible not only tominimize the contact between the combined active ingredients, but also,it is possible to control the release of one of these components in thegastrointestinal tract such that one of these components is not releasedin the stomach but rather is released in the intestines. Anotherembodiment of this invention where oral administration is desiredprovides for combined compounds wherein one of the active ingredients iscoated with a sustained-release material which effects asustained-release throughout the gastrointestinal tract and also servesto minimize physical contact between the combined active ingredients.Furthermore, the sustained-released component can be additionallyenteric coated such that the release of this component occurs only inthe intestine. Still another approach would involve the formulation ofcombined compounds in which the one compound is coated with a sustainedand/or enteric release polymer, and the other compound is also coatedwith a polymer such as a low viscosity grade of hydroxypropylmethylcellulose or other appropriate materials as known in the art, inorder to further separate the active components. The polymer coatingserves to form an additional barrier to interaction with the othercomponent.

[0058] Dosage forms of the combination products of the present inventionwherein one active ingredient is enteric coated can be in the form oftablets such that the enteric-coated compound and the other activeingredient are blended together and then compressed into a tablet orsuch that the enteric coated component is compressed into one tabletlayer and the other active ingredient is compressed into an additionallayer. Optionally, in order to further separate the two layers, one ormore placebo layers may be present such that the placebo layer isbetween the layers of active ingredients. In addition, dosage forms ofthe present invention can be in the form of capsules wherein one activeingredient is compressed into a tablet or in the form of a plurality ofmicrotablets, particles, granules or non-perils, which are then entericcoated. These enteric coated microtablets, particles, granules ornon-perils are then placed into a capsule or compressed into a capsulealong with a granulation of the other active ingredient.

[0059] These as well as other ways of minimizing contact between thecombined compounds, whether administered in a single dosage form oradministered in separate forms but at the same time or concurrently bythe same manner, will be readily apparent to those skilled in the art,based on the present disclosure.

Combination Therapy

[0060] Each therapeutic compound of this invention can independently bein any dosage form, such as those described above, and can also beadministered in various ways, as described above. For example, thecompounds may be formulated together, in a single dosage unit (that is,combined together in one capsule, tablet, powder, or liquid, etc.) as acombination product. Alternatively, when not formulated together in asingle dosage unit, an individual GPIIb/IIIa antagonist may beadministered at the same time as either aspirin, a low molecular weightheparin or TPA, or sequentially, in any order thereof. When notadministered at the same time, preferably the separate administrationsof the compounds occur less than about one hour apart.

[0061] Preferably, the route of administration of therapeuticcombinations herein is by subcutaneous injection. Although it isgenerally preferred that the compounds are administered by the sameroute of administration, it is not necessary for this to be so. Forexample, when a GPIIb/IIIa antagonist and aspirin are administered as atherapeutic combination, it may be preferable for the antagonist to beadministered intravenously and aspirin to be administered orally.

[0062] As is appreciated by a medical practitioner skilled in the art,the dosage of the combination therapy of the invention may varydepending upon various factors such as the pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration, the age, health and weight of the recipient, the natureand extent of the symptoms, the kind of concurrent treatment, thefrequency of treatment, and the effect desired, as described above.

[0063] The proper dosage of a GPIIb/IIIa antagonist and aspirincombination, a GPIIb/IIIa antagonist and low molecular weight heparincombination, or a TPA compound B combination, is readily ascertainableby a medical practitioner skilled in the art, based upon the presentdisclosure. By way of general guidance, typically a daily dosage may beabout 0.01 milligram to about 1 gram of each component. By way ofgeneral guidance, when the compounds are administered in combination,the dosage amount of each component may be reduced by about 70-80%relative to the usual dosage of the component when it is administeredalone as a single agent for the treatment of thrombosis, in view of thesynergistic effect of the combination.

[0064] Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

EXAMPLES

[0065] The invention can be understood further by the followingexamples. Other features of the invention will become apparent to thoseskilled in the art during the following description and exemplaryembodiments that are given for illustration and are not intended to belimiting thereof. Saline (0.9 weight % NaCl) is the vehicle in allexamples.

Example 1 The Combination of Aspirin and a GPIIb/IIIa Antagonist

[0066] Rabbits were anesthetized with ketamine (50 mg/kg i.m.) andxylazine (10 mg/kg i.m.) and then surgically prepared with arterial andvenous catheters. An electromagnetic flow probe was placed on a segmentof an isolated carotid artery to monitor blood flow. Thrombus formationwas induced by electrical stimulation of the carotid artery for 3 min at4 mA using an external stainless-steel bipolar electrode. Carotid bloodflow was measured continuously over a 90-min period to monitor thrombusocclusion. Test agents were infused intravenously 1 hour prior to theelectrical stimulation of the carotid artery and continuously during the90-min period.

[0067] As shown in FIG. 3, following the electrical stimulation,thrombus formation was induced and carotid blood flow was graduallydeclined in saline vehicle-treated animals. At about 40 min afterstimulation, the artery was totally occluded and blood flow was zero.Aspirin at 1 mg/kg/hr i.v. (concentration in saline was 0.167 mg/ml) orCompound (A) (a GPIIb/IIIa antagonist) at 0.03 mg/kg/hr i.v.(concentration in saline was 0.005 mg/ml) did not prevent the occlusionof the artery; and blood flow in these animals was decreased to zero atabout the same time as those in vehicle-treated animals. Surprisingly,Compound (A) 0.03 mg/kg/hr i.v. in combination with aspirin at 1mg/kg/hr i.v. prevented the artery from occlusion and maintained theblood flow at a level of 60-70% of the control level for greater than 90min. These results indicate that a combination of Compound (A) andaspirin at their subtherapeutic doses unexpectedly produced asignificant antithrombotic effect in a rabbit model of arterialthrombosis.

Example 2 The Combination of Dalteparin (Fragmin) and a GPIIb/IIIaAntagonist

[0068] The same experimental protocol as described in Example 1 was usedin this study. As shown in FIG. 4, following the electrical stimulation,thrombus formation was induced and carotid blood flow was graduallydeclined in saline vehicle-treated animals. At about 40 min afterstimulation, the artery was totally occluded and blood flow was zero.Fragmin at 60 U/kg/hr i.v. (concentration in saline was 10 U/ml) was notvery effective in increasing the blood flow. Compound (A) (a GPIIb/IIIaantagonist ) at 0.03 mg/kg/hr i.v. (concentration in saline was 0.005mg/ml) was not effective and blood flow in these animals was decreasedto zero at about the same time as those in saline vehicle-treatedanimals. Surprisingly, Compound (A) at 0.03 mg/kg/hr i.v. in combinationwith dalteparin (fragmin) at 60 U/kg/hr i.v. prevented the artery fromocclusion and maintained the blood flow at a level of 60-70% of thecontrol level for greater than 90 min. These results indicate that acombination of Compound (A) and dalteparin (fragmin) at theirsubtherapeutic doses unexpectedly produced a significant antithromboticeffect in a rabbit model of arterial thrombosis.

Example 3 The Combination of Tissue Plasminogen Activator and aGPIIb/IIIa Antagonist

[0069] Experiments were conducted in rats anesthetized with ketamine(110 mg/kg i.m.) and xylazine (10 mg/kg i.m.). The protocol is similarto the rabbit protocol as described above in Example 1 except that thefree acid of a compound of the formula (B):

[0070] or the free acid of sibrafiban and/or TPA were given 5 min aftera preformed clot was formed. The measured parameters were time toreperfusion, duration of patency and incidence of reocclusion. The dosesof TPA, sibrafiban and compound B used were 1, 0.3 and 0.3 mg/kg i.v.,respectively. The doses of sibrafiban and compound B used in this studywere equal effective in preventing thrombosis in this rat model.

[0071] As shown in Table 1 below, a combination of compound B and TPAproduced unexpectedly better thrombolytic effect than the combination ofsibrafiban and TPA. This result suggests that compound B is a promisinguseful adjunctive agent, which accelerates thrombolysis induced by TPAor other thrombolytic agents. In addition, this result also shows thatcompound B exerted a better enhancement of the thrombolysis induced by asubtherapeutic dose of TPA than sibrafiban. TABLE 1 Sibrafiban +Compound B + TPA TPA TPA (n = 17) (n = 14) (n = 14) Time to 32 ± 6 30 ±4 16 ± 2* reperfusion (min) Duration of 29 ± 2 63 ± 6* 84 ± 2*, **patency (%) Incidence of 15/17 6/14* 1/14*, ** reocclusion

Example 4 Computerized Thromboelastography (FIG. 5)

[0072] Clot formation was monitored at 37° C. in an oscillating plasticcylindrical cuvette (“cup”) and a coaxially suspended stationary piston(“pin”) with a 1 mm clearance between the surfaces, using a computerizedThrombelastograph (CTEG Model 3000, Haemoscope, Skokie, Ill.). The cuposcillates 4°45′ (1/12 radian) in either direction every 4.5 seconds,with a 1 second mid-cycle stationary period; resulting in a frequency of0.1 Hz and a maximal shear rate of 0.1 per second. The pin is suspendedby a torsion wire that acts as a torque transducer. With clot formation,fibrin fibrils physically link the cup to the pin and the rotation ofthe cup (Transmitted to the pin) is displayed on-line using anIBM-compatible personal computer and customized software (HaemoscopeCorp., Skokie, Ill.). The torque experienced by the pin (relative to thecup's oscillation) is plotted as a function of time. The amplitude onthe TEG tracing is a measure of the rigidity of the clot; the peakstrength or the shear elastic modulus attained by the clot, G, is afunction of clot rigidity and can be calculated from the maximalamplitude (MA) of the TEG tracing.

[0073] The following parameters were measured from the TEG tracing (FIG.5):

[0074] R, the reaction time (gelation time) represents the latent periodbefore the establishment of a 3-dimensional fibrin gel network (withmeasurable rigidity of amplitude=2 mm).

[0075] Maximum Amplitude (MA, in mm) is the peak rigidity manifested bythe clot.

[0076] Shear elastic modulus or clot strength (G, dynes/cm²) is definedby: G=(5000* MA)/(96-MA) for computerized TEG.

[0077] Blood Sampling: Blood was drawn from consenting volunteers undera protocol approved by the Human Investigations Committee of WilliamBeaumont Hospital. Using the two-syringe method, samples were drawnthrough a 21 gauge butterfly needle and the initial 3 ml blood wasdiscarded. Whole blood (WB) was collected into siliconized Vacutainertubes (Becton Dickinson, Rutherford, NJ containing 3.8% trisodiumcitrate such that a ratio of citrate whole blood of 1:9 (v/v) wasmaintained. TEG was performed within 3 hrs of blood collection.

Example 5 Platelet Contribution to Clot Strength

[0078] To assess the effect of the TF modification on peak clot strength(G) was measured with and without the addition of TF in paired samplesof WB and PRP. To document the contribution of activated platelets tothe elastic modulus of fibrin clots, the effect of platelet number wasmeasured by serially diluting PRP with PPP. The platelet count of eachdilution was measured before performing TEG. Tissue factor-triggered TEGwas performed with increasing concentrations (0, 0.625, 1.25, 2.5, 5 and10 μM) along with DMSO added to the TEG cups such that the final DMSOconcentration in each TEG sample was 0.3% (v/v). The effect ofGPIIb/IIIa blockade on clot strength was studied by adding increasingconcentrations of c7E3 Fab (Abciximab, Centocor, Malvern, Pa.), cyclicpeptide, peptidomimetic and non-peptide GPIIb/IIIa antagonists (DuPont,Wilmington, Del.) and various LMWH to the TEG cup along with CaCl₂ andTF. The Thrombelastograph maximum amplitude (MA) for platelets MA (PLT))was calculated by subtracting the MA from a platelet-poor plasma (PPP)sample MA (PPP) determined in one thromboelastography well from that ofwhole blood MA (WB) run simultaneously in the second thromboelastographywell.

Example 6 Platelet Aggregation

[0079] Agonist-induced platelet aggregation was measured as change in %light transmission of PRP (platelet count 2×105 per μL). For studyingthe effect of LMWH & GPIIb/IIIa antagonists on platelet aggregation,increasing concentrations were added to PRP for 5 minutes after which0.5 IU/ml thrombin was added. The aggregation response was measured asthe maximum response of the increase in light transmission induced bythrombin, using PPP to establish 100% light transmission.

Example 7 Comparative Efficacy Among Different LMWHs in Inhibiting ClotFormation Induced by Different Stimuli in Human Whole Blood UsingThromboelastography Mediators of Clot Retraction Mean (IC₅₀ [μg/mL])±SEM

[0080] TABLE 2 TF 2.5 ng LPS 6.25 μg Xa 0.25 nM Thrombin 0.3 mU LMWHEnoxaparin 0.95 ± 0.39 2.86 ± 0.35 0.52 ± 0.15 1.19 ± 0.13 Tinzaparin 0.49 ± 0.06*  0.94 ± 0.28*  0.15 ± 0.05*  0.33 ± 0.02* Fraxiparin 2.85± 0.83 3.32 ± 0.85 0.98 ± 0.45 0.78 ± 0.21 TFPI ND 0.09 ± 0.01 ND —

[0081] Comparative efficacy among different LMWHs in inhibiting clotformation induced by different stimuli in human whole blood usingThromboelastography: Syngeristic effects of GPIIb/IIIa antagonists:TABLE 3 Thrombin Thrombin + roxifiban LMWH IC₅₀ (μg/mL) IC₅₀ (μ/mL)enoxaparin 1.19 ± 0.13 0.72 ± 0.18 tinzaparin 0.33 ± 0.03 0.15 ± 0.10fraxiparin 0.57 ± 0.11 0.34 ± 0.08 dalteparin 0.52 ± 0.16 0.24 ± 0.09

[0082] Synergy between LMWH and GPIIb/IIIa antagonist roxifiban onthrombin-induced clot retraction in human blood usingthromboelastography: TABLE 4 Thrombin + tinzaparin Thrombin + enoxaparinThrombin (0.1 μg/mL) (0.5 μg/mL) IC₅₀ (μg/mL) IC₅₀ (μg/mL) IC₅₀ (μg/mL)roxifiban 110 ± 20 61 ± 6.1 50 ± 0.5

What is claimed is:
 1. A method for treating a condition selected fromthe group: acute coronary ischemic syndrome, thrombosis,thromboembolism, thrombic occlusion and reclusion, restenosis, transientischemic attack, and first or subsequent thrombotic stroke comprisingadministration of a sub-therapeutic dose of a low molecular weightheparin and a sub-therapeutic dose of a platelet GPIIb/IIIa antagonistto a mammal in need of such treatment.
 2. A method according to claim 1,wherein the platelet GPIIb/IIIa antagonist is roxifiban.
 3. A methodaccording to claim 1, wherein the low molecular weight heparin istinzaparin.
 4. A method according to claim 1, wherein the administrationis subcutaneous.
 5. A pharmaceutical composition comprising a plateletGPIIb/IIIa antagonist and a low molecular weight heparin and apharmaceutically acceptable carrier.
 6. A pharmaceutical compositionaccording to claim 5, wherein the platelet GPIIb/IIIa antagonist isroxifiban.
 7. A pharmaceutical composition according to claim 5, whereinthe low molecular weight heparin is tinzaparin.
 8. A kit comprising aplatelet GPIIb/IIIa antagonist and a low molecular weight heparin forsubcutaneous injection.
 9. A method of treating thrombosis in a mammalcomprising administering to said mammal a combination of: (i) asubtherapeutic dose of GPIIb/IIIa antagonist selected from the groupconsisting of abciximab, eptifibatide, tirofiban, lamifiban,lefradafiban, sibrafiban, orbofiban, xemilofiban, a compound of theformula (A):

and a compound of the formula (B):

and (ii) aspirin or (iii) a low molecular weight heparin selected fromthe group consisting of tinzaparin, certoparin, parnaparin, nadroparin,ardeparin, enoxaparin, reviparin, reviparin and dalteparin.
 10. Themethod of claim 9 wherein the combination of (i) and (ii) or (iii)provides a synergistic effect.
 11. The method of claim 9, wherein thecombination administered is a combination of the GPIIb/IIIa antagonistand a low molecular weight heparin.
 12. The method of claim 11, whereinthe GPIIb/IIIa antagonist is administered in a subtherapeutic amount.13. The method of claim 11, wherein the GPIIb/IIIa antagonist iscompound A or compound B.
 14. The method of claim 11, wherein the lowmolecular weight heparin is dalteparin (fragmin).
 15. The method ofclaim 11, wherein the GPIIb/IIIa antagonist is compound A or compound Band the low molecular weight heparin is dalteparin (fragmin).
 16. Theuse of a combination of: (i) a subtherapeutic dose of GPIIb/IIIaantagonist selected from the group consisting of abciximab,eptifibatide, tirofiban, lamifiban, lefradafiban, sibrafiban, orbofiban,xemilofiban, a compound of the formula (A):

and a compound of the formula (B):

and (ii) aspirin or (iii) a low molecular weight heparin selected fromthe group consisting of tinzaparin, certoparin, parnaparin, nadroparin,ardeparin, enoxaparin, reviparin, fragmin and dalteparin for themanufacture of a medicament for the treatment of thrombosis.
 17. Amethod of treating thrombosis in a mammal comprising administering tosaid mammal (i) tissue plasminogen activator and (ii) a compound of theformula: